A decrease in oxygen flow to an organ, called ischemia, triggers a complex series of events that affect the structure and function of virtually every organelle and subcellular system of the affected cells. Ischemia-reperfusion (resumption of blood flow) injury leads to the production of excessive amounts of reactive oxygen species (ROS) and reactive nitrogen species (RNS), thus causing oxidative stress which results in a series of events such as alterations in mitochondrial oxidative phosphorylation, depletion of ATP, an increase in intracellular calcium and activation of protein kinases, phosphatases, proteases, lipases and nucleases leading to loss of cellular function/integrity. It has been shown that the inflammatory response induced by ischemia followed by reperfusion is largely responsible for tissue and organ damage.
Ischemic-reperfusion injury is a serious problem in organ transplantation because the harvested organ is removed from the body, isolated from a blood source, and thus deprived of oxygen and nutrients for an extended period of time. A critical problem to be addressed in present-day kidney transplantation procedures is the relatively high incidence of delayed graft function (DGF) due to acute tubular necrosis (ATN) following surgery. Illustratively, DGF affects about 20-35% of kidney transplants in many transplant centers and is the most common complication of the immediate post-operative period in renal transplantation. Although the incidence and definition of DGF vary among transplant centers, the consequences most frequently involve a prolonged hospital stay, additional invasive procedures and additional costs to the patient and health-care system. Delay in graft function not only affects the individual patient, it also impacts the infrastructure for organ procurement and sharing as a consequence of the drain it places on the available organ supply. DGF also increases the risk of early acute rejection episodes and increases early graft loss from chronic rejection.
With current preservation methods, cold ischemia resulting from organ preservation has been identified as a major risk factor in causing DGF after transplant. For kidneys, cold ischemia times in excess of 24 hours are associated with a significantly increased risk of DGF. In the mid-1960's, cold preservation of kidneys was effectively achieved by using machine perfusion and a solution derived from cryoprecipitated plasma. Thereafter, by simple cold-storage methods were introduced and involved the use of a cold crystalloid solution. Since the early successes with kidneys, preservation solutions have evolved into entirely synthetic defined media designed to prevent cold ischemic injury by suppression of cell swelling and provision of metabolic support. An early synthetic solution, a lactobionate-based solution (UW) from the University of Wisconsin has been used for both pancreas and liver. With the advent of synthetic and serum-free preservation formulations, the quality and duration of feasible organ preservation have improved. Despite this, however, clinical data on DGF in kidneys and other problems involving renal cell structure and morphology clearly demonstrate that such solutions are not completely successful in preventing ischemic injury or insult.
In addition, acute renal failure (ARF) secondary to ischemic or nephrotoxic injury also remains a common and potentially devastating problem in clinical nephrology, with a persistently high rate of mortality despite significant advances in supportive care. Over several decades, a number of studies have illuminated the roles of persistent vasoconstriction, tubular obstruction, cellular structural and metabolic alterations, and the inflammatory response in the pathogenesis of ARF. Treatments and remedies for ARF have been hampered by the multifaceted response of the kidney to ischemia, as well as a lack of early markers for ARF. Recent advances in cellular and molecular biology of ischemic and nephrotoxic renal injury have shown that proximal tubule cells undergo a complex temporal sequence of events, including loss of cell polarity, cell death due to apoptosis and necrosis, de-differentiation and proliferation of viable cells, and re-establishment of the epithelial phenotype.
As a result of ischemic or nephrotoxic damage, cells may die through two different processes. Apoptosis or programmed cell death (“cell suicide”) is a physiological mechanism for removing senescent, damaged or abnormal cells that affects individual cells. Apoptosis is initiated by an endonuclease and is characterized by DNA fragmentation into multiples of 180-200 base pairs. Apoptotic cells are ingested by macrophages or neighboring cells without release of proteolytic enzymes or toxic oxygen species and the process is not accompanied by inflammation. By contrast, necrosis (“cell murder”) is a pathological process that affects populations of cells and results in focal tissue destruction, inflammation and often serious systemic consequences. Apoptotic cell death has now been shown to play an important role in an increasing array of kidney diseases, including ischemia, ischemia-reperfusion, nephrotoxins, polycystic kidney disease, obstruction, and glomerular diseases. Down-regulation of apoptosis therefore offers a unique and powerful therapeutic approach to the amelioration of several acute and chronic kidney injuries.
An individual is considered to have acute renal failure when the patient's serum creatinine value either (1) increased by at least 0.5 mg/dL when the baseline serum creatinine level was less than 2.0 mg/dL; (2) increased by at least 1.5 mg/dL when the baseline serum creatinine level was greater than or equal to 2.0 mg/dL; or (3) increased by at least 0.5 mg/dL, regardless of the baseline serum creatinine level, as a consequence of exposure to radiographic agents.
cDNA microarray techniques have allowed the identification of neutrophil gelatinase-associated lipocalin (NGAL) as a highly induced transcript in the kidney early after ischemic and nephrotoxic injury. The role of NGAL in the kidney has yet to be elucidated. NGAL is a member of the lipocalin family of proteins and is characterized as a secreted 25 kDa glycoprotein found in granules of human neutrophils. (Kjeldsen et al, 1993, J. Biol. Chem. 268:10425-10432). Lipocalins, which are able to bind small lipophilic substances, share a common three-dimensional β-barrel structure which functions, in at least some lipocalins, to bind lipophilic ligands, e.g., steroid, bilin, retinoid, or other lipid. Murine forms of NGAL (homologs) from mice and rats are known. In mice, NGAL has been designated as NGAL, 24p3 protein, SIP24, P25, lipocalin 2, and uterocalin. NGAL in rats is known as NGAL or alpha 2-microglobulin. A full-length cDNA encoding human NGAL protein has been cloned and sequenced. The human NGAL gene, which includes seven exons and six introns, has also been cloned and sequenced, and its expression in various tissues has been analyzed. The human NGAL gene encodes a polypeptide of 197 amino acids, with a 19- or 20-amino acid signal sequence, and a mature NGAL polypeptide containing 178 amino acids. The motifs Gly-X-Trp (amino acids 48-50 in mature human NGAL) and Thr-Asp/Asn-Tyr (amino acids 132-134 in mature human NGAL) are present in all known lipocalins. On the basis of X-ray crystallography, it has been suggested that these motifs are important in the tertiary β-barrel structure shared among the lipocalins. The cysteine residues 95 and 194 in the human NGAL sequence are conserved, and have been reported to form an intramolecular disulfide bridge. Human NGAL contains a single N-glycosylation site (an asparagine residue) at position 65 of the mature amino acid sequence (approximately position 84 or 85 of the pre-NGAL polypeptide).
A mechanism that may underlie ATN is mis-localized iron. Unbound iron can catalyze the conversion of H2O2 to OH and OH− (the Haber-Weiss reaction) or form reactive ferryl or perferryl species. These ions mutagenize many types of molecules including lipids, nucleotides and the DNA backbone. Catalytic iron, released from free hemoglobin and myoglobin into urine or blood, and peroxidized lipids have been documented in many forms of acute renal failure, including chemotherapy, ischemia-reperfusion, transplant ischemia, and in proteinuria-mediated tubular damage. Preloading animals with iron worsens the disease, and conversely chelating iron with deferoxamine or bacterial siderophores blunts the damage. Iron-catalyzed damage is thought to be one of the earliest events in kidney dysfunction and is likely to be important in other organs, including the heart and the liver (See Mori et al: Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury. J Clin Invest 115:610-621, 2005).
Cells acquire iron from carrier proteins (such as transferrin) and by cell surface iron transporters (such as divalent metal transporter I). Intracellular iron is controlled by the actions of the iron responsive proteins (such as IRP1, IRP2), the ferritin complex and heme oxygenase I. Because IRPs are modulated by hypoxia, oxidative stress, and phosphorylation, changes in their activity may play an important role in ischemic disease, by regulating formation of ferritin complexes, which protect cells from iron mediated damage. Ferritin is an iron-phosphorous-protein complex, comprising approximately 23% iron, formed in the intestinal mucosa. Ferritin is the storage form of iron in tissues such as liver, spleen, and bone marrow. Hemoglobin and myoglobin molecules in blood and muscle, respectively, require iron-binding to catalyze transfer of oxygen to cells. However, few other aspects of iron trafficking, storage or metabolism are known in ischemic cells or in other types of tissue damage, despite the primacy of catalytic iron in their pathogenesis.
Despite the many pathways of producing ATN, a number of investigators have discovered general underlying mechanisms of proximal tubule cell damage. These have included the release of cytokines. One idea is that ischemic cells and tubular toxins such as free myoglobin and hemoglobin produce high concentrations of iron locally in the nephron. It is thought that this iron is catalytically active, and produces oxygen radicals. Evidence that iron catalyzed cell damage is pathogenic and leads to proximal tubular dysfunction includes the finding that tubular damage is blunted by infusions of iron chelators. Additional support for the idea that iron is central to the mechanism of organ dysfunction after ischemia comes from experiments that used iron free-bacterially derived, iron chelators, called siderophores, to blunt the effects of ischemia-reperfusion injury in an in vitro model of cardiac ischemia. Each of these general mechanisms is thought to be the principle pathogenic event during different stages of ATN. Iron catalyzed damage is thought to be one of the earliest events in kidney dysfunction.
It is currently unknown how the proximal tubule captures NGAL. Indeed an unambiguous identification of receptors for most lipocalins is still lacking. Perhaps megalin, which is necessary for reclamation of RBP, is also the NGAL receptor (Christensen et al., 1999, J Am Soc Nephrol. 10(4) 685-95). In fact, knockout of megalin leads to the appearance of NGAL in the urine, but these animals were also, unexpectedly, found to have much higher levels of NGAL message (Hilpert et al., 2002, Kidney Int. 62(5)1672-81), suggesting that urinary NGAL might have derived from local synthesis rather than a failure to capture the filtered load. Despite this ambiguity, NGAL is similar to other lipocalins, such as RBP and α-2u globulin lipocalin (see Borghoff et al., 1990, Annu Rev Pharmacol Toxicol. 30:349-67), which enter the cell by a megalin pathway and traffic to lysosomes for degradation. These data contrast with the trafficking of NGAL in cell lines that do not express megalin (such as embryonic kidney cells) and where the protein escapes degradation (see Yang et al., 2002, Mol. Cell 10(5):1045-56). Similarly, transferrin is also degraded after delivery to lysosomes by a megalin-cubulin based pathway in the proximal tubule (see Kozyraki et al., 2001, Proc. Natl Acad Sci USA 98(22)12491-6), whereas it usually recycles in cell lines. Hence it is reasonable to propose that after filtration, NGAL is captured by megalin and degraded by the proximal tubule and is not recycled. This hypothesis is supported by the observation that full length NGAL does not reappear in the blood at delayed time points post-injection.
There remains a need for compositions and methods suitable for preventing, reducing, or ameliorating ischemic injury, e.g., cold ischemic injury, in organs such as the kidney. Such compositions would be useful both in treating a patient's original organs, as well as organs used for transplantation. Also needed are new biomarkers that can be used to detect toxic damage to cells, for example nephrotoxicity, in patients following drug administration. New and improved methods of treating and reducing ischemic-reperfusion injury to tissues and organs caused by organ transplantation, and of treating and reducing structural and metabolic alterations of organ cells, are clearly useful and important to practitioners and patients alike.